JP3295424B2 - Detergent enzymes - Google Patents

Abstract

PCT No. PCT/DK92/00273 Sec. 371 Date Feb. 8, 1994 Sec. 102(e) Date Feb. 8, 1994 PCT Filed Sep. 11, 1992 PCT Pub. No. WO93/05134 PCT Pub. Date Mar. 18, 1993.This inventions is in the field of detergent enzymes. More specifically, the invention relates to the use of proteases from fungi of the genus Verticillium for detergent purposes.

Description

Description: TECHNICAL FIELD The present invention is in the field of detergent enzymes. More particularly, the invention relates to the use of proteases from fungi of the genus Verticillium for detergent purposes.

Background Art Fungi belonging to the genus Verticillium are well known in the literature and they are known to be pathogenic to insects and plants. Fungi are also known to produce proteolytic enzymes that have been studied for fungal pathogenicity.

Summary of the Invention Now, surprisingly, the fungus Verticillium
illium) members have been found to have excellent cleaning performance.

Thus, the present invention relates to strains CBS NO145.70; CBS NO.146.70; CBS
NO.247.68; and a protease obtainable from any of CBS NO.464.88, or strain CBS NO145.70; CBS NO.14
6.70; CBS NO.247.68; and a detergent composition comprising a protease having the same or partially the same immunochemical properties as the protease derived from any of CBS NO.

In another aspect, the invention relates to Verticillium.
illium) or a mutant thereof or a member thereof.

In yet another aspect, the invention relates to the strain CBS NO145.70; CBS N
O.146.70; protease obtainable from any of CBS NO.247.68; and CBS NO.464.88, or strain CBS NO145.
70; CBS NO.146.70; CBS NO.247.68; and CBS NO.464.88
The present invention provides a detergent additive comprising a protease having the same or partially the same immunochemical properties as the protease derived from any of the above.

In another aspect, the invention relates to Verticillium.
illium) or a mutant thereof or a member of a mutant thereof.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be further described with reference to the accompanying drawings.

FIG. 1 shows the relationship between the proteolytic activity (relative%) of the enzyme of the present invention and the temperature (° C.) (Δ is pH 9.5; □ is 0.1% ST
At pH 9.5 with the addition of PP).

FIG. 2 shows the proteolytic activity (relative%) and pH of the enzyme of the present invention.
Shows the relationship.

Detailed Disclosure of the Invention The present invention comprises a detergent composition obtainable from a fungal strain of the genus Verticillium. Fungi belonging to the genus Verticillium are well known and have been described in the literature. Verticillium (Ve
strains have been deposited and are available from various international depositaries, for example, CBS NO.247.68; CBS NO.145.70; CBS
NO.146.70; or CBS NO.464.88.

Proteases can be obtained by known methods and are described in the literature, e.g., by culturing in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts, followed by recovery of the enzyme of interest. Or can be produced by using recombinant DNA engineering.

Proteases In the context of the present invention, a suitable protease is a strain of Verticillium, or a protease obtainable from a mutant or variant thereof, or a strain of Verticillium, for example, A protease having the same or partially the same immunological properties as a protease obtainable from a strain of V. bulbillosum.

An enzyme mutant or mutant enzyme refers to an enzyme that can be obtained by modifying the DNA nucleotide sequence of a parent gene or a derivative thereof. Enzyme variants, as well as mutant enzymes, are expressed and produced upon insertion of the DNA nucleotide sequence encoding the enzyme into a suitable vector in a suitable host organism. The host organism need not necessarily be the same as the organism from which the parent gene is derived.

Immunochemical properties can be measured immunologically by a cross-reactivity identity test. The identity test is performed by the well-known Octellurony double immunodiffusion method or by NH Axelsen; Handb.
ook of Immunoprecipitation-in-Gel Techniques; Blackwell Scientific Publishing (1983), 5
It can be performed by double-headed cross immunoelectrophoresis according to Chapters 14 and 14.

Detergent Composition The detergent composition of the present invention can include one or more surfactants, which can be of the anionic, nonionic, cationic, amphoteric or zwitterionic type, or mixtures thereof. . Typical examples of anionic surfactants are linear alkyl benzene sulphonate (LAS); alkyl sulphate (AS); alpha olefin sulphate (AOS); alkoxy ethoxy sulphate and alkali metal salts of natural fatty acids. Examples of nonionic surfactants are alkyl polyethylene glycol ether; nonylphenol polyethylene glycol
Ethers; fatty acid esters of sucrose and glucose; polyethoxylated alkyl glucosides.

Detergent compositions of the present invention or other detergent components known to those skilled in the art, such as bleach, bleach activators, corrosion inhibitors, sequestrants, anti-soil-redeposition agents, fragrances, enzymes and bleaches. Agents, foam enhancers, chelating agents, fillers, fabric softeners and the like. The composition of the present invention comprises J. Forbes (Folbe, J; Surfactants in Con
sumer Products.Theory, Technology and Application;
Springer Fellark 1987, especially "Frame form
ulations for liquid / powder heavy-duty detergent
(see section entitled "s").

It is presently believed that the detergent composition of the present invention may contain an enzyme preparation in an amount corresponding to 0.0005-0.5 CPU of protease per wash.

The detergent compositions of the present invention can be formulated into any conventional form, for example, as powders, liquids, and the like.

The detergent composition of the present invention advantageously comprises one or more other enzymes, such as lipase; amylase; cellulase; oxidase; and / or peroxidase, and proteases of other origin, usually in the detergent composition. Can be contained.

The proteases of the present invention can be included in the detergent composition by adding a separate additive containing a detergent protease or by adding a combined additive comprising different detergent enzymes.

The additives of the present invention, i.e., separate or combined additives, can be formulated, for example, as granules, liquids, slurries. Preferred formulations of detergent additives are dust-free granulates, liquids, especially stabilized liquids, slurries or protected enzymes. Dust-free granules are UK patent 1,362,365
Or it can be manufactured according to U.S. Patent 4,106,991,
If desired, it can be coated by methods known to those skilled in the art. The detergent enzyme can be mixed before or after granulation. Liquid enzyme preparations can be stabilized, for example, by adding a polyol such as propylene glycol; a sugar or a sugar alcohol; lactic acid or boric acid according to established methods.
Other enzyme stabilizers are known to those skilled in the art. Protected enzymes can be prepared according to the methods described in European Patent Application 238,216.

The present invention is further described in the following examples, which are not intended to limit the scope of the invention as claimed in any way.

Example 1 Production Example Verticillium b.
ulbillosum), CBS NO.247.68 with the following composition (
500m baffle plate containing 100m medium with
On a rotating vibrating table in an Erlenmeyer flask with
Incubated at 25 ° C: Sucrose 100g Soybean flour 40g Na_TwoHPO_Four× 12H_TwoO 10g Pluronic ) Stabilize 0.1g medium by heating at 120 ° C for 45 minutes.

After incubation for 12 days, the proteolytic activity of the 21 CPU / culture was measured using the method described below.

After separation of the solid material, the protease was purified by a conventional chromatography method.

The yield of one culture broth is 30 m with 270 CPU /
Met. Purity was greater than 90% as determined by SDS-PAGE.

Strain V.bulbillosum, CBS NO.145.70;
Vulbulillosum, CBS NO.146.70; and V.
A preparation from such a scala sporium (suchlasporium) var. Scala sporium, CBS NO. 464.88 was obtained in a similar manner.

Example 2 Properties of the Enzyme The preparation prepared according to Example 1 was subjected to the following properties.

 The molecular weight of 30 kD was measured by SDS-PAGE. LKB Am
Holin (Ampholine ) Isoelectric focusing on PAG plate
Measured a pI higher than 9.3. Protease activity
The properties are as follows: PMSF, α-1-antitrypsin (antitrypsi
n) and turkey-inhibited by egg white proteinase inhibitor
Harmed. EDTA and soy-protein inhibitors are
Has no effect on the activity of the enzyme.

The relationship between temperature and activity was measured using casein as a substrate. The analysis for proteolytic activity described above was performed with modifications where the incubation temperature was varied within the interval of 15 ° C to 70 ° C. The enzyme has proteolytic activity from below 15 ° C to above 70 ° C and has an optimum temperature in the range of 45-60 ° C; around 55 ° C.

The dependence of the activity on pH was determined by a similar procedure, using a buffer adjusted to a predetermined pH value in the pH range from 6 to 11. The results are shown in FIG. The enzyme has proteolytic activity at pH values from less than 6 to more than 11, and has an optimum pH in the range of pH 8 to pH 11.

Analysis for Proteolytic Activity Proteolytic activity is measured using casein as a substrate. One casein protease unit (CPU) is defined as the amount of enzyme that releases 1 mM primary amino per minute (measured by comparison with the serine standard method) upon incubation under standard conditions, ie, 25 ° C. and PH9.5 for 30 minutes. Measured. The folded printed matter AF228, which describes the analytical method,
It is available upon request from Novo Nordisk A / S (Denmark), which is incorporated herein by reference.

Example 3 Two sets of cleaning performance tests were performed isothermally at 20 ° C. on cotton soiled with grass juice for 10 minutes.

In the first set, 2.0 g / g of commercial American type powder detergent was used. The detergent was dissolved in about 6 dH (German hardness) water. pH was 9.5. Table 1 shows the test results.

In the second set, 2.0 g with bleach and activator
A commercial American type powder detergent was used.
The detergent was dissolved in about 6 dH (German hardness) water. pH is 9.
It was five. Table 2 shows the test results.

In both sets, the fiber / wash ratio is 6 g of fiber per wash.
Met.

Following washing, the fabric was rinsed in running tap water and air dried. The specific reflectance (% R) was measured at 460 nm.

As a measure of the washing performance, the differential regular reflectance, ΔR, was used, which was equal to the value obtained by subtracting the regular reflectance after washing without adding the enzyme from the regular reflectance after washing with the addition of the enzyme.

Continuation of the front page (72) Inventor: Dambman, Klaus, Denmark, 2860 Sabork, Denmark. Te. Ho. 61 (72) Inventor Patkar, Sharmkant Anant, Denmark-2800 Ringbee, Denmark, Christophers Alle 91 (56) References Can J Microbiol. , Vol. 43, No. 3, p227-233, 1997 FEMS Microbiol Lett. , Vol. 126, No. 3, p227-231, 1995 (58) Fields investigated (Int. Cl. ⁷ , DB name) C11D 3/386 C12N 9/58 C12R 1: 645 CAOLD (STN) CAPLUS (STN) REGISTRY (STN) WPIDS ( STN) PubMed

Claims (11)

(57) [Claims] 1. The following properties: (1) has a molecular weight of 30 kDa as measured by SDS-PAGE; (2) has proteolytic activity from below 15 ° C. to above 70 ° C .; (3) exhibits proteolytic activity from a pH lower than 6 to a pH higher than 11, and has an optimum pH in the range of pH 8 to pH 11; A detergent composition comprising one or more proteases, which can be obtained from a microorganism belonging to the genus Verticillium. [Claim 2] CBS No. 145.70, CBS No. 146.70, CBS No. 2
The detergent composition according to claim 1, comprising one or more proteases obtainable from 47.68 or CBS No. 464.88 or a mutant or mutant thereof. 3. The detergent composition according to claim 1, further comprising one or more other enzymes. 4. The detergent composition according to claim 3, wherein said other enzyme is amylase, lipase, cellulase, oxidase and / or peroxidase. 5. The following properties: (1) have a molecular weight of 30 kDa as determined by SDS-PAGE; (2) have proteolytic activity from below 15 ° C. to above 70 ° C .; (3) exhibits proteolytic activity from a pH lower than 6 to a pH higher than 11, and has an optimum pH in the range of pH 8 to pH 11; Detergent additive comprising one or more proteases, which can be obtained from a microorganism belonging to the genus Verticillium. (6) CBS No. 145.70, CBS No. 146.70, CBS No. 2
Detergent additive according to claim 5, comprising one or more proteases obtainable from strain 47.68 or CBS No. 464.88 or a mutant or mutant thereof. 7. The detergent additive according to claim 5, further comprising one or more other enzymes. 8. The detergent additive according to claim 7, wherein said other enzyme is amylase, lipase, cellulase, oxidase and / or peroxidase. 9. A detergent additive according to claim 5, which is provided in the form of granules, liquids or protected enzymes. 10. The detergent additive according to claim 9, wherein the granules are dust-free granules. 11. The fluid of claim 9, wherein said fluid is a stabilizing liquid.
9. The additive for detergents according to item 1.